Anti human il 10 apc

PBMCs (1 × 10⁶) were stained with CD4 PerCP, CD25 Alexa488, and FoxP3 PE for the identification of Treg populations (BD FoxP3 staining protocol, according to the manufacturer’s instructions). CD4 PerCP, CD25 Alexa488, CD27 APC, and CD45RA PE-Cy7 (eBioscience) were used to assess surface phenotype. In some experiments, CD39 APC, PD1 PE-Cy7, and glucocorticoid-induced tumor necrosis factor receptor (GITR) APC (Biolegend) were analyzed on Treg populations. Intracellular cytokine staining was performed to determine IFN-γ and IL-10 production at the single-cell level as described previously using anti-human IFN-γ-PE-Cy7 and anti-human IL-10 APC (both from Biolegend). For the evaluation of TGF-β secretion, the surface expression of the latency-associated peptide (LAP) protein (anti-human LAP-PE-Cy7, Biolegend) was assessed. For all flow cytometry experiments, sample acquisition and analysis were carried out on a FACSCanto flow cytometer using the BD FACSDiva software (BD Biosciences). Negative control samples were incubated with irrelevant, isotype-matched mAbs in parallel with experimental samples.

Flow cytometry experiments were performed to assess T cells co-cultured with MenSCs. The fluorochrome-conjugated antibodies included Alexa Fluor 647 anti-FoxP3 (BD Biosciences, USA), FITC anti-human IFN-γ, PerCP/Cy5.5 anti-CD4, APC anti-human IL-10, and PE anti-CD25 (All from Biolegend, USA) were employed. Antibodies were used at the concentrations recommended by the manufacturers. For assessment of cytokines, cells were treated with 50 µg/mL PMA (Sigma, USA), 1 µg/mL ionomycin (Sigma, USA) and 0.7 µg/mL Monensin (BD Biosciences, USA) 6 h. before staining procedure. For intracellular staining of Foxp3 and cytokines, transcription factor buffer set (BD Biosciences, USA) was used for cell permeabilization and fixation according to the manufacturer’s instruction. Cells were also stained with Live/Dead fixable near red fluorescent dye (Molecular Probes, USA) to separate the alive and dead populations before antibody-specific gating. The stained cell was then read by flow cytometer Attune NxT flow cytometer and analyzed by FlowJo software (FlowJo, LLC, USA).